Biomarkers, 2010; 15(7): 625–638
ORIGINAL ARTICLE
Potential of the hepatic transcriptome e xpression profile of the striped seabream (Lithognathus mormyrus) as an environmental biomarker Yana Yudkovski1, Andreja Ramšak2, Meirav Ausland1, and Moshe Tom1 Israel Oceanographic and Limnological Research, Haifa, Israel, and 2National Institute of Biology, Marine Biology Station, Piran, Slovenia
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Abstract The potential of the hepatic transcriptome expression profile evaluated in a sentinel feral fish to serve as an environmental biomarker was examined. Expression profiles of Lithognathus mormyrus individuals were exhibited using cDNA microarray and were related to the set of exposure conditions at their sites and dates of collection. Expression profiles of individual fish were reasonably clustered according to the fish samples. In addition, several sample-specific gene clusters were determined, designated sample gene signatures. The selection procedure for future optimal reference RNA is discussed. The relationship between transcriptome expression and fish samples indicated a potential for using the former as an environmental biomarker. Keywords: Aquatic organisms; environmental pollution/ecotoxicology; gene expression; biomonitoring; fish; cDNA microarray
Introduction Anthropogenic impact on natural biota is a major environmental threat and its monitoring is essential for designing and adjusting environmental policy. Sentinel organisms inhabiting an examined habitat are exposed to, and affected by, their surroundings. Consequently, pollution-affected biological parameters measured in suitable organs of a sentinel species have been widely suggested as biomarkers of pollution effects (Moore et al. 2004). The subject biomarker in the present study is the transcriptome expression profile evaluated in a sentinel fish. It is a complex parameter composed of the list of relative expression levels of specific genes in an individual specimen or in a defined group of individuals, in comparison to the corresponding levels in a reference transcriptome. The term, expression profile, as used in this study is exactly defined in the Materials and methods section below. Due to its comprehensiveness, this biomarker was postulated to respond sensitively to a wide range of environmental threats.
The potential of evaluating coordinated expression of genes for the detection of the biological effects of environmental pollutants has been discussed in recent literature applying microarray platforms as a detection tool (Ankley et al. 2006, Carvan et al. 2008). Comparability over a broad time scale and wide geographical range is considered an essential requirement for an environmental biomarker and can be achieved by utilizing a common reference RNA. The best theoretical reference is a mRNA mixture taken from individuals that have never been exposed to pollution, more tightly relating gene differential expression in the sampled fish to pollution effect. A feral fish, the striped seabream, Lithognathus mormyrus was chosen as a sentinel species of Mediterranean coastal habitats (Yudkovski et al. 2008). Itisa demersal fish,inhabiting the Israeli coast of the Mediterranean in both polluted and pristine sandy habitats. It interacts with the bottom through its bottom-feeding habits and burial behaviour and its gut usually contains sediment engulfed with ingested food (Froglia 1977, Kallianiotis et al. 2005). It is a protandrous
Address for Correspondence: Moshe Tom, Israel Oceanographic and Limnological Research, P.O.B 8030, Haifa, 31080, Israel. Tel: 972-4-8565-257. Fax: 972-4-8511-911. E-mail:
[email protected] (Received 11 April 2010; revised 08 July 2010; accepted 16 July 2010) ISSN 1354-750X print/ISSN 1366-5804 online © 2010 Informa UK, Ltd. DOI: 10.3109/1354750X.2010.510579
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626 Yana Yudkovski et al. hermaphrodite whose immature gonad is an ovotestis, either developing into a testis or an ovary. However, the developed gonad always includes residual tissue of the opposite sex. L. mormyrus reaches maturity during its third year (Besseau & Brusle-Sicard 1995) and it exhibits no external sex-distinguishing characters. Hence, it is not possible to determine sex outside the reproduction season. The sex reversal from male to female occurs in a wide size range. Hence, it is difficult to determine the sex of an individual within this range, from its size (Kallianiotis et al. 2005, Bauchot & Hureau 1986, Kraljevic et al. 1995). The reproduction season extends from June to August–September in the Northern hemisphere. As in other vertebrates, fish liver is commonly involved in xenobiotic detoxification. Hence, studies using L. mormyrus as a sentinel fish have adopted quantitative reverse transcription–real-time polymerase chain reaction (qRT-PCR) for evaluation of the hepatic expression of the biomarker genes. These genes included metallothionein, cytochrome P4501A, vitellogenin and zona radiata protein (ZRP), modified to include appropriate specific transcript standards compatible with the target fish (Yudkovski et al. 2008, Funkenstein et al. 2004). In addition, a cDNA microarray was constructed of transcripts cloned from multipollutant-affected L. mormyrus livers (Auslander et al. 2008). Microarray platforms have been used to study the effects of pollutants on multigene expression in fish, mainly through controlled laboratory exposure to pollutants (Carvan et al. 2008). In addition, one field study compared transcriptome expression profiles in different populations of the sentinel European flounder (Platichthys flesus) exposed to assumedly different sets of environmental exposure conditions (Falciani et al.2008). The term set of environmental exposure conditions, as used in this study applies to the comprehensive, not necessarily completely defined set, of environmentalconditions prevailing in the sampled site at the date of sampling. Ideally, the fulfilment of three working hypotheses would make the present spotted hepatic cDNA assemblage of L. mormyrus an efficient tool to test the relationships between expression profiles of individual fish and sets of environmental conditions. These hypotheses are: (1) mutual exposure history to pollutants of all individual fish composing a sample, due to their permanent residence in the examined habitat; these fish are assumed to be characterized by similar phenotype and genotype; (2) bias of the hepatic transcript assemblage spotted on the microarray toward pollution-affected genes; (3) different pollution status of the two sampled sites, one being exposed to more severe pollution pressure than the other. In the context of the present sentinel and the sampled habitats, only indirect and circumstantial evidence exists
to validate the depicted hypotheses. Hence, permanent residence in the examined habitat was postulated based on the L. mormyrus demersal biology. Similar phenotypes were judged by similar size and reproductive activity, and no genetic background data were available. Bias of the utilized cDNA assemblage toward pollutionaffected genes was deduced from the methodology of its construction (Auslander et al. 2008). Evidence indicating different pollution status of the two sampled sites is presented and discussed in the Materials and methods section. Therefore, a top-down analytical approach was implemented. It started from relating the characteristics of the gene expression profiles of individual fish to the fish samples exposed to the poorly defined site- and time-related sets of environmental exposure conditions. Later, the compatibility of the working hypotheses with the revealed relationships between fish samples and individual gene expression profiles was explored. Valid hypotheses are expected to lead to more uniform withinsamples expression profile characteristics than among samples, affected mainly by the mutual exposure of each sample to a specific set of environment exposure conditions. This study and similar future studies, using other species in a variety of habitats, is the first step towards establishing the examined species as an environmental sentinel.
Materials and methods Fish sampling, habitat characterization and organ processing Fish were caught alive by gillnetting in February–March 2007 and 2008 at two sites on the Mediterranean coast of Israel, Haifa (35.00°E 32.83°N) and Dor (34.91°E 32.62°N). The sampling plan was aimed at examining changes in the hepatic expression profiles at different dates and sites of collection, assuming that both parameters may affect the pollution pressure. Winter sampling was conducted to avoid individuals during their reproduction season. The Haifa site samples of 2007 and 2008 were designated H7 and H8, respectively, and the corresponding Dor samples were designated D7 and D8. The Haifa site is located in the southern part of Haifa Bay, just outside the Haifa harbour, near the Haifa urban and industrial region, and in close proximity to the polluted Kishon estuary. The Dor site is located 25 km south of the Haifa site, remote from urban and industrial regions, bordering a marine nature reserve. This circumstantial evidence for different anthropogenic impact at the two sites was further indicated by the levels of mercury and cadmium in the sediment of both sites. The levels of mercury and cadmium at Dor were estimated from
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Transcriptome expression as an environmental biomarker 627 the corresponding levels at the Taninim site, located 9 km south of Dor, at the same water depth. Like Dor, it is remote from urban and industrial regions and the closest available source of pollution data, postulated to represent fairly the Dor site. The 2005 heavy metal evaluation in the sediment of the Haifa and Taninim (34.89°E 32.54°N) sites (Herut et al. 2006) revealed 0.270–0.919 and 0.061–0.270 μg g−1 dry weight mercury levels and 0.08–1.54 and 0.04–0.07 μg g−1 dry weight cadmium levels in the sediments of the two sites, respectively. It addition, evaluation of the hepatic cytochrome P4501A transcript levels in L. mormyrus sampled at the Haifa and Dor sites in 2004–5 revealed sevenfold higher levels in the former site (Yudkovski et al. 2008). Hence, the two sites can be reasonably postulated to be under different pollution impacts, Dor being the cleaner of the two. Fish were sacrificed by decapitation 3 h after sampling, followed by immediately dissecting their liver and gonads. The livers were divided into smaller aliquots, snap-frozen in liquid nitrogen, and stored at -80°C for several days, prior to RNA extraction. Gonads were fixed in 4% formaldehyde and their reproductive activity was determined by microscopic observation of fixed gonads smeared between two slides, measuring the average ova diameter of 20 oocytes. Ten reproductively arrested individuals were selected for each sample, totalling 40 fish. The biological characteristics of the samples are presented in Table 1 revealing statistically significant uniform fish weight and length among the four ten-fish samples (oneway ANOVA, df=3, F(weight)=0.357, F(length)=0.195, p 0.5 M>0.5 Dq 850753 dq 850968 dq 850969 dq 849664 dq 849734 NA dq 849848 dq 850207 dq 850861 D -0.54 -0.51 DQ 849911 Apolipoprotein 14kDa Fish lipid metabolism D M0.05). CYP1A revealed significant differences (df=2, F=6.144 and p
